Antibody Prurification Methods
Antibody purification is a multistep process by which contaminants of source is removed and antibody with high purity is obtained. Antibodies are widely used as
injectables and parenteral products for human use. Monoclonal antibodies
dominate the antibody field. Before going to the antibody purification process, let’s
see the contaminants of the antibody source.
Basic Structure of an
Antibody
Antibody is a multi-chain protein, secreted in order to
fight with the antigens which enters the body and thereby preventing possible
infections. Generally an antibody is a polypeptide of four chains, having two
identical light chains and two identical heavy chains. Antibodies are
glycoproteins.
Antibodies are produced in mice, rabbits, etc. Nowadays
antibodies are expressed in cell culture with good yield. When antibodies are
taken from animal source possible contaminants could be serum proteins such as
albumin, transferrins and cell degradation products like DNA and cellular
proteins. Currently serum free media for cell culture are developed which can
ease the antibody purification process. With a combination of chromatographic
steps and precipitation methods one can purify the antibody with good yield and
resolution.
Purification Methods
The choice of a purification method is based on a these factors:
- Nature of antibody,
- Nature of feedstock,
- Scale of production,
- Economics – cost and other factors,
- Process Timings, and
- Desired purity.
- Sample Preparation
- Capture
- Initial Purification
- Secondary Purification
- Polishing / Formulation
Let’s go in detail on each of these steps for better
understanding,
Antibody
Purification: Step 1: Sample Preparation
Sample prep or sample preparation is the initial step in
which crude protein sample is conditioned or making it ready for the initial
capture step. Generally this step involves changing pH or Ionic strength,
dilution of the crude sample or addition of salts for the ionic strength. These
above mentioned techniques may increase the cost in manufacturing so they may
not be feasible in the large scale production process. So what is suitable in
large scale antibody purification process is to use buffer exchange by size
exclusion chromatography or to use ultrafiltration or diafiltration. Dialysis
is one common method followed in lab scale level but in production scale it is
not feasible.
either by centrifugation or Filtration, sometimes both the methods are combined
to get faster results.
Antibody precipitation can be done to precipitate out, salts
used for this purpose include ammonium sulphate, Poly ethylene glycol, etc. if
the antibody is expressed in cell line media contaminants (dye – phenol red)
need to be removed which in-turn can bind to the column and reduce the
efficiency of the purification process.
Purification: Step 2: Capture
Capturing is the first major purification step in a process
typically involves binding the antibody to chromatographic matrix, while
impurities either flow through or are differentially eluted from the column. The
process need to be optimized for better results, good yield and purity.
widely used for antibody purification.
- Immunoaffinity
- Immobilized Metal Affinity Chromatograhy (IMAC)
- Ion – Exchange Chromatograhy (IEC)
- Hydrophobic Interaction Chromatography (HIC)
- Hydroxyapatite
- Size – Exclusion chromatography (SEC)
All other techniques, except Hydroxyapatite chromatographic technique,
are explained in the previous posts, so let’s look into the Hydroxyapatite
chromatography.
In hyroxyapatite chromatography, interaction of the antibody
with calcium and phosphate has a major role to play. Mostly elution is done
using a phosphate buffer gradient. As this technique has the ability to bind to
DNA and separate out idiotypes. Hydroxyapatite chromatography is used mostly in
lab scale because only low flow rates can be used in this technique and resin
cannot be reused. Due to these drawback hydroxyapatite chromatodraphic
application is limited to lab scale purification.
Antibody
Purification: Step 3: Secondary Purification
The secondary purification step is selected based on the
nature and the optimization requirement of the crude antibody source, the
initial capture step can often give purities in the range of 80 to 95%. However, for higher purity grades in excess of 99% secondary purification is
required. In addition to protein contaminants, other impurities such as DNA,
endotoxins, viruses, and aggregates need to be removed. In such cases, a
multistep procedure is almost inevitable. All the same techniques used for
initial capture can be used for secondary purification. Indeed, many of the techniques,
such as HIC and Hydroxyapatite chromatography, are used more often used as polishing
steps than as initial capture steps.
Purification: Step 4: Polishing /
Formulation
Final polishing /
formulation step can be considered as a part of purification in which it removes
conditions that would impair the stability or utility of the antibody in its
intended use. As the downstream processing rule, more no of steps results in
more loss of the product. Final formulation may be as simple as a straightforward
sterilization by membrane filtration through a sterile filter with pores 0.2 lm
or less. Another relatively simple formulation step is adjusting the antibody
concentration, either by dilution with buffer or by concentration on ultra
filtration. In other circumstances, the buffer composition may need to be
changed to achieve optimal stability of the antibody. For this purpose, SEC or
diafiltration is widely used. A more complex formulation step would be the addition
of excipients to confer stability. Finally, the antibody solution may need to be
lyophilized/ Freeze dried to confer stability, and the liquid formulation may
be changed to be compatible with the lyophilization process.
better yield, resolution and better stability.
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